Thirty-five-color Spectral Flow Cytometry Panel for Total and Phosphorylated Protein Status, and Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood

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Raw flow cytometry data using human peripheral blood mononuclear cells.

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STRATEGY FOR FLUOROCHROME SELECTION AND FLUOROCHROME CHARACTERIZATION The first step for the panel development consisted of identifying the best possible 35 fluorochrome combination. Based on the criteria, 35 fluorochromes were selected (Supplemental Figures 2A, B), minimizing fluorochrome pairs with very high similarity indices and providing the lowest complexity index (complexity index=35). The distribution of the selected fluorochromes across lasers and emission wavelengths is presented in Supplemental Table 2. The pairs with the highest similarity indices, and hence higher predicted spread between them, were BB515 and Alexa Fluor 488 (0.98), BV421 and Super Bright 436 (0.97), Super Bright 436 and eFluor 450 (0.94), and Alexa Fluor 647 and Spark NIR 685 (0.92). Additionally, an effort was made to minimize the use of unconjugated reagents which needs to be conjugated by ourselves. Out of the 35 fluorochromes used in this panel, only one: TBK1 (Cell signaling technology, Danvers, MA) was unconjugated antibody and conjugated by PE/Cy5® Conjugation Kit - Lightning-Link® (abcam, Boston, MA). Then, spread was assessed by calculating the SSM using FlowJo™ version 10.8.2 with the same data used to rank fluorochrome brightness (Supplemental Figure 2C). As expected, based on the spectrum and similarity indices, the pairs with the highest SSM values were PE-Cy5 and Alexa Fluor 647 (SSM=34), BB515 and Alexa Fluor 488 (SSM=25), PE-Cy5 and PE Fire540 (SSM=25), PE-Cy5 and APC (SSM=18), and PE-Cy5 and Spark NIR 685 (SSM=15) (Supplemental Figure 2C). Of the 1,190 combinations of fluorochromes, 12 had an SSM greater than 10 and SSM greater than 5 were only 38 which the majority (97%) having SSM values of 5 or less. PANEL DESIGN STRATEGY Our goal of this project was to develop a panel allowing for the identification of all principal leukocyte subsets in human peripheral blood mononuclear cells blood (PBMC) including some intracellular cell activation markers using a minimum number of markers. The rationale behind was to offer the research community a most broadly applicable platform for in-depth phenotyping of human PBMC using 5-laser spectral flow instruments (UV/violet/blue/green/red) widely available for researchers and clinicians. In the process of designing this Optimized Multicolor Immunofluorescence Panel (OMIP), we carefully assorted the individual markers and always followed the goal of covering all principal leukocyte subsets in human PBMC, that is T cells, B cells, monocytes, dendritic cells (DCs), and natural killer (NK) cells.

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